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Subjects

Abstract

A long-standing objective in neuroscience has been to image distributed neuronal activity in freely behaving animals. Here we introduce NeuBtracker, a tracking microscope for simultaneous imaging of neuronal activity and behavior of freely swimming fluorescent reporter fish. We showcase the value of NeuBtracker for screening neurostimulants with respect to their combined neuronal and behavioral effects and for determining spontaneous and stimulus-induced spatiotemporal patterns of neuronal activation during naturalistic behavior.

Acknowledgements

We thank K. Kawakami and A. Muto (National Institute of Genetics (Japan)) for sharing tg(gSA2AzGFF49A;UAS:GCaMP7a) and tg(gSA2AzGFF152B), here referred to as GR152:Gal4, which was provided to us by R. Portugues and L. Knogler (Max Planck Institute of Neurobiology), K. Asakawa (National Institute of Genetics (Japan)) for providing tg(mnGFF7;UAS:eGFP), J. Ngai (University of California, Berkeley) for tg(OMP4; UAS:GCaMP1.6), M. Ahrens (Janelia Research Campus) for sharing tg(HuC:H2B-GCaMP6s), H. Baier and K. Slanchev (Max Planck Institute of Neurobiology) for sharing tg(HuC:Gal4;UAS:eGFP) and tg(HuC:Gal4;UAS:GCaMP6s), and L. Godinho (Technical University of Munich) for providing tg(HuC:Gal4;UAS:GCaMP6s). We are grateful to J. Huisken and F. Fahrbach for providing assistance on building the SPIM; J. Fuchs assisted with the SPIM setup and experiment. We thank B. Wolfrum, P. Rinklin, J. Rebling, D. Razansky and the central machine shop of Helmholtz Zentrum Muenchen for help with the design and production of the arenas; H. Rolbieski for administrative support and help with data organization; T. Durovic and J. Ninkovic for assistance with the two-photon microscopy experiments; M. Fabiszak, C. Penningroth and L. Garrett for valuable comments on the manuscript. We are grateful for support from the European Research Council under grant agreements ERC-StG: 311552 (G.G.W., A.L., P.S.) and the Helmholtz Alliance ICEMED (G.G.W.).

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Contributions

P.S. designed and implemented NeuBtracker with important contributions from H.J., M.K. and A.C.; programmed control software; conducted neurobehavioral imaging experiments including SPIM; analyzed data; and wrote the manuscript. A.L. designed and implemented neurobehavioral and two-photon experiments, conducted p-ERK imaging experiments, analyzed data, created illustrations and wrote the manuscript. A.S. and T.L. implemented image registration routines and improved tracking algorithm and analyzed data together with S.S. M.C. carried out the first implementation of tracking algorithm and performed original image and signal processing. C.C.P. carried out the initial implementation of SPIM. A.M. conducted photoaversion experiments on immobilized fish. A.S. was responsible for animal husbandry and provided support for neurobehavioral imaging. S.R. generated tg(HuC:GCaMP6s). W.W. and V.N. supported the project and provided feedback on the manuscript. G.G.W. conceived and supervised the work, analyzed data and wrote the manuscript.